FIGURE 5.

Differential activity of recombinant human TDP1 for single-stranded DNA substrates harboring 5′-phosphotyrosine (Y19) and 5′-fluorescein (6-FAM19). A, scheme for the processing pathways of Y19 (left) and 6-FAM19 (right) by TDP1. Because of the nucleosidase activity of TDP1 (19), which removes the last 3′-end base, the Y19 substrate can be processed in two ways. One is that the 5′-tyrosine is removed first (P19) before the 3′-terminal adenine (P18) (clockwise). The other is that the 3′-adenine is removed first (Y18) before the 5′-tyrosine (P18) (counterclockwise). An asterisk indicates the radiolabeled site. B, each substrate was incubated with serial dilutions (1:3) of TDP1 at 25 °C for 30 min with the highest TDP1 concentration starting at 1 μm. Processing of 6-FAM19 by TDP1 yielded one product (right), whereas processing of Y19 yielded two distinct products (left), indicating that TDP1 processes 5′-phosphotyrosine but not 5′-fluorescein.